重组纤溶酶原激活剂及其突变体的表达研究

Expression of the recombinant plasminogen activator (rPA) and its mutants

利用PCR技术 ,获得了人体组织型纤溶酶原激活剂tPAcDNA的缺失突变体———rPA ;在此基础上 ,运用定点突变技术 ,得到rPA的定点突变体———rPA(KHRR2 96～ 2 99AAAA) ,rPA(A473S)和二者的复合突变体rPA(KHRR2 96～2 99AAAA A473S) ;将rPA及其 3个突变体分别亚克隆至原核表达载体pET 2 8a(+)中 ,获得表达载体pErA ,pErA(K) ,pErA(A)和pErA(KA) .酶切鉴定和序列分析结果均表明实现了实验设计的氨基酸突变 .表达载体转化大肠杆菌 ,经IPTG诱导、菌体裂解及SDS PAGE电泳分析发现 ,只缺失而无突变的pErA和突变的pErA(A)都未表达目的蛋白 ,突变体pErA(K)与pErA(KA)则获高水平表达 ;蛋白产量分别占菌体总蛋白的 35 .97%与 37.71% ,分子量均为 39.6kD .Westernblotting显示 ,表达产物与抗tPA抗体呈特异性阳性反应 .该产物经初步纯化后进行复性与活性 (mFAPA)测定 ,结果表明其复性产物具有明显的体外纤溶活性 .以上结果为rPA突变体的进一步纯化。

rPA, the deleted mutant of tPA cDNA, was obtained by the PCR technology. Then, the rPA was mutated by substituting the KHRR with AAAA at positions 296-299 (rPA(K)), the A with S at position 473 (rPA(A)) and at both positions(rPA(KA)) on the same DNA, respectively, with site-directed mutagenesis. That four were subcloned into the prokaryotic expression vector pET-28a(+) separately and the constructed vectors pErA, pErA(K), pErA(A) and pErA(KA) were characterized with restriction enzyme indigesting and sequence analysing. All of them showed that the expected variants were acquired. After they were transformed into E.coli strain BL21(DE3) and induced by IPTG, the lysates of the bacteria body were separated on SDS-PAGE. The result indicated that the expression of mutants pErA(K) and pErA(KA) were achieved, but pErA and pErA(A) were not expressed under the same conditions. The expressed proteins of interest, whose molecular weight were 39.6?kD,were in accordance with those of the estimated and their magnitude make up 35.97% and 37.71% of the total proteins of E.coli separately.They had a specific reactivity with anti-tPA antibodies as shown by Western blotting analysis. After primarily purified and refoldded, the expressed proteins displayed relatively high fibrin lysis activity in vitro on modified Fibrin Agarose Plate Assay (mFAPA). All above laid a foundation for further purification, activity assay in vivo and large scale production.